虎杖中一种新的2-吡喃酮合酶基因的克隆和功能研究（英文）
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摘要
4-羟基-6-甲基-2-吡喃酮(2-吡喃酮)及其衍生物是一类重要的植物次生代谢产物,具有抗虫、抗真菌等功能,在工业上可用于生产可再生化学平台间苯三酚和1,3,5-三氨基-2,4,6-三硝基苯. 2-吡喃酮合酶(2PS),一种Ⅲ型聚酮合酶(PKSs),是合成2-吡喃酮的关键酶.本研究以中药材虎杖(Polygonum cuspidatum Sieb. et Zucc)为材料,从中分离鉴定了一种新的2-吡喃酮合酶(Pc2PS). Pc2PS与已知的几种2PSs的氨基酸序列相似性为54%～56%.通过体外酶促反应鉴定功能发现,Pc2PS可以催化1分子乙酰-CoA与2分子丙二酰-CoA,缩合生成4-羟基-6-甲基-2-吡喃酮;也可以只利用3分子丙二酰-CoA,以相同的效率缩合生成2-吡喃酮.由此可以看出,乙酰-CoA存在与否并不影响该酶的催化效率.随后,我们测定了Pc2PS以丙二酰-CoA为单一底物时的酶动力学参数.虽然之前报道的2PSs也可以只利用丙二酰-CoA生成2-吡喃酮,但与Pc2PS不同的是,乙酰-CoA的缺失会大大降低催化效率.另外,对Pc2PS基因的组织表达特异性检测结果表明,该基因主要在虎杖根中表达,在叶中的表达量很低.本研究丰富了2PS的种类,并为2-吡喃酮的生物合成提供了基因资源.
Derivatives of 4-hydroxy-6-methyl-2-pyrone(2-pyrone) are actively involved in plant resistance to insects and pathogens, and are considered as potential renewable chemical platforms for phloroglucinol and 1,3,5-triamino-2,4,6-trinitrobenzene.2-Pyrone synthase(2 PS), one of the type Ⅲ polyketide synthases(PKSs), is the key enzyme to produce 2-pyrone. In this study, a novel Pc2PS was isolated from Polygonum cuspidatum Sieb. et Zucc., a traditional Chinese herb used for the treatment of cough,suppurative conditions, and hypertension. The Pc2PS shared 54%–56% amino acid sequence identity with 2 PSs of other species.Enzymatic activity analysis showed that Pc2PS could catalyze the condensation of one molecule of acetyl-CoA with two molecules of malonyl-CoA at almost the same efficiency as that of three molecules of malonyl-CoA alone for production of 4-hydroxy-6-methyl-2-pyrone. Thus, the presence or absence of acetyl-CoA in the reaction mixture did not affect catalytic efficiency. This catalytic property of Pc2PS differs from that of previously reported Gh2 PSs, which catalyzed malonyl-CoA condensation to form the same products as Pc2PS but at a lower rate in the absence of acetyl-CoA. In addition, we determined the kinetics of Pc2PS with malonyl-CoA alone as the substrate for the first time. Pc2PS showed tissue-specific expression in P. cuspidatum; Pc2PS was predominantly expressed in the root, whereas few transcripts were detected in the leaf. This study enriches the known diversity of2 PS proteins, and provides a novel genetic resource for 2-pyrone biosynthesis.
Derivatives of 4-hydroxy-6-methyl-2-pyrone(2-pyrone) are actively involved in plant resistance to insects and pathogens, and are considered as potential renewable chemical platforms for phloroglucinol and 1,3,5-triamino-2,4,6-trinitrobenzene.2-Pyrone synthase(2 PS), one of the type Ⅲ polyketide synthases(PKSs), is the key enzyme to produce 2-pyrone. In this study, a novel Pc2PS was isolated from Polygonum cuspidatum Sieb. et Zucc., a traditional Chinese herb used for the treatment of cough,suppurative conditions, and hypertension. The Pc2PS shared 54%–56% amino acid sequence identity with 2 PSs of other species.Enzymatic activity analysis showed that Pc2PS could catalyze the condensation of one molecule of acetyl-CoA with two molecules of malonyl-CoA at almost the same efficiency as that of three molecules of malonyl-CoA alone for production of 4-hydroxy-6-methyl-2-pyrone. Thus, the presence or absence of acetyl-CoA in the reaction mixture did not affect catalytic efficiency. This catalytic property of Pc2PS differs from that of previously reported Gh2 PSs, which catalyzed malonyl-CoA condensation to form the same products as Pc2PS but at a lower rate in the absence of acetyl-CoA. In addition, we determined the kinetics of Pc2PS with malonyl-CoA alone as the substrate for the first time. Pc2PS showed tissue-specific expression in P. cuspidatum; Pc2PS was predominantly expressed in the root, whereas few transcripts were detected in the leaf. This study enriches the known diversity of2 PS proteins, and provides a novel genetic resource for 2-pyrone biosynthesis.
引文
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[16]Ferrer J L,Jez J M,Bowman M E,et al.Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis.Nat Struct Biol,1999,6(8):775-784
[17]Abe I,Utsumi Y,Oguro S,et al.A plant type III polyketide synthase that produces pentaketide chromone.J Am Chem Soc,2005,127(5):1362-1363
[18]Taura F,Iijima M,Yamanaka E,et al.A novel class of plant type IIIpolyketide synthase involved in orsellinic acid biosynthesis from Rhododendron dauricum.Front Plant Sci,2016,7(1452):1-15
[19]Ma L Q,Guo Y W,Gao D Y,et al.Identification of a Polygonum cuspidatum three-intron gene encoding a type III polyketide synthase producing both naringenin and p-hydroxybenzalacetone.Planta,2009,229(5):1077-1086
[20]Abe I,Takahashi Y,Morita H,et al.Benzalacetone synthase.Anovel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum.Eur JBiochem,2001,268(11):3354-3359
[21]Raiber S,Schroder G,Schroder J.Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine(Pinus strobus).A single Arg/His difference determines the activity and the pH dependence of the enzymes.FEBS Lett,1995,361(2-3):299-302
[22]Wang X,Zhang Z,Dong X,et al.Identification and functional characterization of three type III polyketide synthases from Aquilaria sinensis calli.Biochem Biophys Res Commun,2017,486(4):1040-1047
[23]Li L,Aslam M,Rabbi F,et al.PpORS,an ancient type IIIpolyketide synthase,is required for integrity of leaf cuticle and resistance to dehydration in the moss,Physcomitrella patens.Planta,2017,247(2):527-541
[24]Abe I,Oguro S,Utsumi Y,et al.Engineered biosynthesis of plant polyketides:chain length control in an octaketide-producing plant type III polyketide synthase.J Am Chem Soc,2005,127(36):12709-12716
[25]Zhou W,Zhuang Y,Bai Y,et al.Biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose.Microb Cell Fact,2016,15(1):149
[2]Keating T A,Walsh C T.Initiation,elongation,and termination strategies in polyketide and polypeptide antibiotic biosynthesis.Curr Opin Chem Biol,1999,3(5):598-606
[3]Schroder J.A family of plant-specific polyketide synthases:facts and predictions.Trends Plant Sci,1997,2(10):373-378
[4]Helariutta Y,Elomaa P,Kotilainen M,et al.Chalcone synthaselike genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida(Asteraceae).Plant Mol Biol,1995,28(1):47-60
[5]Eckermann S,Schroder G,Schmidt J,et al.New pathway to polyketides in plants.Nature,1998,396(6709):387-390
[6]Jez J M,Austin M B,Ferrer J,et al.Structural control of polyketide formation in plant-specific polyketide synthases.Chem Biol,2000,7(12):919-930
[7]Pietiainen M,Kontturi J,Paasela T,et al.Two polyketide synthases are necessary for 4-hydroxy-5-methylcoumarin biosynthesis in Gerbera hybrida.Plant J,2016,87(6):548-558
[8]Peng W,Qin R,Li X,et al.Botany,phytochemistry,pharmacology,and potential application of Polygonum cuspidatum Sieb.et Zucc.:a review.J Ethnopharmacol,2013,148(3):729-745
[9]Guo Y-W,Guo H-L,Li X,et al.Two type III polyketide synthases from Polygonum cuspidatum:gene structure,evolutionary route and metabolites.Plant Biotechnol Rep,2013,7(3):371-381
[10]Ma L Q,Pang X B,Shen H Y,et al.A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum.Planta,2009,229(3):457-469
[11]Beuerle T,Pichersky E.Enzymatic synthesis and purification of aromatic coenzyme a esters.Anal Biochem,2002,302(2):305-312
[12]Wang J,Wang X H,Liu X,et al.Synthesis of unnatural 2-substituted quinolones and 1,3-diketones by a member of typeⅢpolyketide synthases from Huperzia serrata.Org Lett,2016,18(15):3550-3553
[13]Larkin M A,Blackshields G,Brown N P,et al.Clustal W and Clustal X version 2.0.Bioinformatics,2007,23(21):2947-2948
[14]Kumar S,Stecher G,Tamura K.MEGA7:molecular evolutionary genetics analysis version 7.0 for bigger datasets.Mol Biol Evol,2016,33(7):1870-1874
[15]Zhang H,Guo Y-W,Yang Y-D,et al.Cloning of a novel type IIIpolyketide synthase encoded by a three-intron gene from Polygonum cuspidatum.J Plant Biochem Bio,2012,23(1):104-111
[16]Ferrer J L,Jez J M,Bowman M E,et al.Structure of chalcone synthase and the molecular basis of plant polyketide biosynthesis.Nat Struct Biol,1999,6(8):775-784
[17]Abe I,Utsumi Y,Oguro S,et al.A plant type III polyketide synthase that produces pentaketide chromone.J Am Chem Soc,2005,127(5):1362-1363
[18]Taura F,Iijima M,Yamanaka E,et al.A novel class of plant type IIIpolyketide synthase involved in orsellinic acid biosynthesis from Rhododendron dauricum.Front Plant Sci,2016,7(1452):1-15
[19]Ma L Q,Guo Y W,Gao D Y,et al.Identification of a Polygonum cuspidatum three-intron gene encoding a type III polyketide synthase producing both naringenin and p-hydroxybenzalacetone.Planta,2009,229(5):1077-1086
[20]Abe I,Takahashi Y,Morita H,et al.Benzalacetone synthase.Anovel polyketide synthase that plays a crucial role in the biosynthesis of phenylbutanones in Rheum palmatum.Eur JBiochem,2001,268(11):3354-3359
[21]Raiber S,Schroder G,Schroder J.Molecular and enzymatic characterization of two stilbene synthases from Eastern white pine(Pinus strobus).A single Arg/His difference determines the activity and the pH dependence of the enzymes.FEBS Lett,1995,361(2-3):299-302
[22]Wang X,Zhang Z,Dong X,et al.Identification and functional characterization of three type III polyketide synthases from Aquilaria sinensis calli.Biochem Biophys Res Commun,2017,486(4):1040-1047
[23]Li L,Aslam M,Rabbi F,et al.PpORS,an ancient type IIIpolyketide synthase,is required for integrity of leaf cuticle and resistance to dehydration in the moss,Physcomitrella patens.Planta,2017,247(2):527-541
[24]Abe I,Oguro S,Utsumi Y,et al.Engineered biosynthesis of plant polyketides:chain length control in an octaketide-producing plant type III polyketide synthase.J Am Chem Soc,2005,127(36):12709-12716
[25]Zhou W,Zhuang Y,Bai Y,et al.Biosynthesis of phlorisovalerophenone and 4-hydroxy-6-isobutyl-2-pyrone in Escherichia coli from glucose.Microb Cell Fact,2016,15(1):149